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The Gram stain was devised by the Danish physician, Hans Christian Gram, while working in Berlin in 1883. He later published this procedure in 1884. At the time, Dr. Gram was studying lung tissue sections from patients who had died of pneumonia.

In his paper, Dr. Gram described how he was able to visualize what we now call Staphylococcus, Streptococcus, Bacillus, and Clostridiain various histological sections. Interestingly, Dr. Gram did not actually use safranin as a counter stain in the original procedure (Gram negative cells would be colorless). He instead recommended using Bismarck brown as a counter stain to enable tissue cell nuclei to be visualized.

A method of staining bacteria using a violet stain. The gram staining characteristics (denoted as positive or negative). A heat fixed bacterial smear is stained with crystal violet (methyl violet), treated with 3% iodine/potassium iodide solution, washed with alcohol and counterstained. The method differentiates bacteria into two main classes, gram-positive and gram-negative.

There are four basic steps of the Gram stain, which include applying a primary stain (crystal violet)or Methyl violet to a heat-fixed smear of a bacterial culture, followed by the addition of a mordant (Gram's iodine), rapid decolorization with alcohol or acetone, and counterstaining with Safranin or basic fuchsin.

First prepare your slide. You do this by placing bacteria on a slide in a drop of water, allowing them to dry and then heat fixing them. Heating the slide kills the bacteria and makes sure that the bacteria a stuck to the slide and wont wash away during the staining procedure

Several alcohols have been studied, and it has been reported that the more complex the alcohol, the slower the decolorization action. As the carbon chain lengthens, decolorization is slower. Kisskalt (84) found decolorization power decreasing in the following order: methyl, ethyl, propyl, butyl, and amyl alcohol. Conn found a mixture of equal parts of methyl and isopropyl alcohols to have very similar decolorization properties to ethyl alcohol. In practice, however, no known advantage can be gained by substituting the higher alcohols for ethyl alcohol.

After the counterstain has been rinsed off, the slide is placed between some absorbent paper and the excess water gently blotted off. Care must be taken not to rub the slide with the blotting paper because this would remove the adhering bacteria.

Gram stained preparations have to be observed with bright-field optics. Phase-contrast microscopy does not allow the recognition of true colours. Gram-positive bacteria may be seen under phase-contrast as red cells. Using bright-field optics, Gram-positive cells are purple or blue and Gram-negative pinkdue to counter stain with Safranin..

We know that the Gram stain is not an all-or-nothing phenomenon, but that quantitative variations in Gram-positivity exist between different species, and within the same species during different parts of the growth cycle or under different environmental conditions. We know that only intact cells are Gram-positive, so that cells which are even gently broken become Gram-negative. We know that bacterial protoplasts, devoid of cell wall, are still Gram-positive, indicating that it is probably the semipermeable membrane which is somehow involved in the reaction.

Gram’s staining method is not without its problems. It is , complicated, and prone to operator error. The method also requires a large number of cells However, it is also central to phenotypic microbial identification techniques.

The Gram staining procedure does not always give clear-cut results. Some organisms are Gram-variable and may appear either Gram-negative or Gram-positive according to the conditions. With these types of organisms, Gram-positive and Gram-negative cells may be present within the same preparation

Some Gram-positive bacteria appear Gram-negative when they have reached a certain age, varying from a few hours to a few days. On the other hand, some Gram-negative bacteria may become Gram-positive in older cultures. For this reason it is strongly recommended to use very young cultures for the staining procedure, after growth has become just visible.

It is necessary that it is stained at two or three different ages (very young cultures should be used). If an organism changes from positive to negative or vice versa during its growth cycle, this change should be recorded with a statement as to the age of the culture when the change was first observed. In case a Gram-variable reaction is observed it is also good to check the purity of the culture.

The genera Actinomyctes, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. The staining of these organisms result in an uneven or granular appearance

Since the original procedure of Gram, many variations of the Gram staining technique have been published. Some of them have improved the method, others include some minor technical variants of no value. Bartholomew (1962) has pointed out that each variation in the Gram staining procedure has a definite limit to its acceptability.

There is no gram procedure which can be referred to as the best for all laboratories and for all situations. It is recommended that the young microbiologists adopt at least two of the well-accepted methods, practice them until he is familiar with their characteristics,

Although it may seem strange, the reason why bacteria with these two major types of bacteria cell walls react differently with Gram's stain appears to be unconnected with the wall structure itself. The exact mechanism of the staining reaction is not fully understood, however, this does not detract from its usefulness.

For slide libraries and teaching collections that will be stored for longer periods, immersion oil can be removed with xylene solution and the slides can be cover slipped using Per mount to prevent fading.

Even new molecular methodologies typically take hours rather than minutes. " This simple staining procedure remains the most useful test performed in the microbiology lab. Results from a Gram's stain can tell volumes about an infection within 15 minutes of a specimen's arrival in the lab, while most other microbiology results require 24 hours or more.